46 Chapter 3 Quantitative real-time PCR (qRT-PCR) reactions were performed in triplicate on two biological replicates (see above) of all cell lines included in the MET antibody validation panel using: TaqMan® Gene Expression Assays against MET and HPRT1 (Hs01565584_m1, Hs02800695_m1; Life Technologies™/Applied Biosystems®), TaqMan® Universal Master Mix II, no UNG (Life Technologies™/Applied Biosystems®) and the 7500 Fast Real-Time PCR System (Life Technologies™/Applied Biosystems®) according to the manufacturer’s protocol for 25 μL reaction volumes. To determine MET mRNA expression levels, a threshold (0.2) was set that falls in the exponential phase (4347825 Rev. F; Applied Biosystems®) of both target (MET) and endogenous control (HPRT1) across all samples. Subsequently, within sample normalization of MET expression was performed according to the relative standard curve method (4371095 Rev A; Applied Biosystems®). Protein isolation and western blot analysis Cells were cultured in T75 flasks until 75-90% confluence was reached and harvested in lysis buffer (1% SDS, 10mM Tris, pH 7.5). Protein concentrations of the lysates were measured using the Pierce® BCA Protein Assay Kit (Thermo Scientific; Bleiswijk, The Netherlands). Western blot analysis was performed on the Mini Protean II system (Bio-Rad; Veenendaal, The Netherlands). Cell extracts, mixed with 2x Laemmli Sample Buffer (Bio-Rad) including 2-mercaptoethanol, containing 18 μg of protein were separated on a 4-20% gradient polyacrylamide gel (Thermo Scientific). Subsequently, the protein fragments were transferred during 1.5 hour onto a PVDF membrane (GE Healthcare; Eindhoven, The Netherlands). After washing, the membrane was blocked for 1 hour with powder milk (5%; Royal FrieslandCampina; Amersfoort, the Netherlands) dissolved in PBS-Tween 0.1%. Next, the membranes were incubated O/N at 4°C with the primary MET antibodies (Supplementary table S3). After washing multiple times with PBS-Tween 0.1%, the membranes were incubated for 1hr with the secondary ECL™ Anti-Rabbit IgG, HRP-Linked Whole Ab (1:5000; NA934V; GE Healthcare). After intensive washing with PBS-Tween 0.1%, SuperSignal® West Femto Maximum Sensitivity Substrate (34095; Thermo Scientific) was used for detection. Beta-actin was visualized as loading control for each sample by means of the same protocol, using the primary mouse monoclonal antibody against beta-actin (1:1000; clone AC-74; Sigma-Aldrich®) and the secondary Anti-Mouse IgG, HRP-linked whole Ab GPR (1:20000; NXA931; GE Healthcare). Immunocytochemical staining of MET antibody validation cell line panel Cells were cultured on Nunc™ Lab-Tek™ Chamber slides (Thermo Scientific) until 60-90% confluence was reached. Subsequently, the cells were washed with cold
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